The present invention relates to the production of recombinant bovine pancreatic desoxyribonuclease I in methylotrophic yeast. In particular, the present invention relates to a bovine pancreatic desoxyribonuclease I that is secreted by the methylotrophic yeast into the growth medium from which it is purified. Also provided are use and kits of the recombinant bovine pancreatic desoxyribonuclease I.
Bovine pancreatic desoxyribonuclease I is an industrial product with a wide range of applications. For instance, applications in the field of cell biology make use of bovine pancreatic desoxyribonuclease I in standard laboratory procedures for the purpose of dissociating cell tissue and isolating single cells. Such processes are always accompanied by rupture and lysis of some cells. As a consequence, DNA is released from these cells into the intercellular space and/or the dissociation medium and causes unwanted cell clumping. Adding bovine pancreatic desoxyribonuclease I to the dissociation medium is a preferred means to prevent unwanted cell clumping as the enzyme has been shown not to be cytotoxic in concentrations of up to 1 mg/ml. Thus, cell clumping is prevented by hydrolysing DNA. For purposes of tissue culture, bovine pancreatic desoxyribonuclease I is also used in combination with other enzymes such as collagenase or trypsin (Kaighn, M. E., In: Tissue culture, methods and applications; Kruse, P. F. & Patterson, M. K., eds., Academic Press, New York & London, 1973, 54–58).
Regarding the fields of molecular biology and nucleic acid biochemistry, bovine pancreatic desoxyribonuclease I is used in applications such as nick translation, the production of random DNA fragments, desoxyribonuclease I protection assays such as transcription factor footprinting, removal of DNA template after in vitro transcription, removal of DNA from RNA samples prior to applications such as RT-PCR, and removal of DNA from other preparations generated by biological and/or biochemical procedures, to name but a few (Sambrook, Fritsch & Maniatis, Molecular Cloning, A Laboratory Manual, 3rd edition, CSHL Press, 2001). Thus, removal of DNA is effected by hydrolysing DNA.
DNase I is also used in medical applications aimed at reducing the viscoelasticity of pulmonary secretions (Liao, T. H., J. Formos. Med. Assoc. 96 (1997) 481–487). Whereas for this particular purpose desoxyribonuclease I of human origin is the preferred enzyme, desoxyribonuclease I from bovine pancreas is preferred for the different kinds of research use exemplified above. Thus, bovine pancreatic desoxyribonuclease I is an enzyme that is produced in an industrial scale and sold as a regular industrial product (e.g. desoxyribonuclease I from bovine pancreas EC 3.1.21.1, Item No. 1284932 in the 2002 catalogue of Roche Diagnostics GmbH, Mannheim).
Bovine pancreatic desoxyribonuclease I has a molecular weight of about 30,000 daltons and an enzymatic activity optimum at pH 7.8. Bovine pancreatic desoxyribonuclease I hydrolyses phosphodiester linkages of DNA, preferentially adjacent to a pyrimidine nucleotide yielding DNA molecules with a free hydroxyl group at the 3′ position and a phosphate group at the 5′ position. The average chain length of a limit digest is a tetranucleotide. There are four desoxyribonucleases derived from bovine pancreas which are all glycoproteins. They differ from each other either in a carbohydrate side chain or polypeptide component. Bovine pancreatic desoxyribonuclease I has diverse chemical activity acting upon single stranded DNA, double stranded DNA and chromatin (Liao, T. H., Mol. Cell Biochem. 34 (1981) 15–22). Similarly to other desoxyribonucleases, bovine pancreatic desoxyribonuclease I appears to be modulated in vivo by actin which is taking the effect as a cellular inhibitor (Lazarides, E., and Lindberg, U., Proc. Natl. Acad. Sci. USA 71 (1974) 4742). Moreover, like other desoxyribonucleases, bovine pancreatic desoxyribonuclease I is activated by divalent metal ions. Maximum activation is attained with Mg2+ and Ca2+. A metallosubstrate, such as a magnesium salt of DNA is necessary. Citrate completely inhibits magnesium-activated but not manganese-activated desoxyribonuclease I. Desoxyribonuclease I is inhibited by chelating agents such as EDTA, and by sodium dodecyl sulfate (Sambrook, Fritsch & Maniatis, Molecular Cloning, A Laboratory Manual, 3rd edition, CSHL Press, 2001).
When desoxyribonuclease activity is quantified, the present document refers to “units”. Thus, the nucleolytic activity of bovine pancreatic desoxyribonuclease I is quantified using a photometric assay similar to Kunitz, M. (J. Gen. Physiol. 33 (1950) 349–62 and 363). The “specific desoxyribonuclease activity” or “specific activity” of a given preparation is defined as the number of units per mg of potein in the preperation, determined by the method described in detail in Example 12.
A “methylotrophic yeast” is defined as a yeast that is capable of utilising methanol as its carbon source. The term also comprises laboratory strains thereof. In case a methylotrophic yeast strain is auxotrophic and because of this needs to be supplemented with an auxiliary carbon-containing substance such as, e.g. histidine in the case of a methylotrophic yeast strain unable to synthesise this amino acid in sufficient amounts, this auxiliary substance is regarded as a nutrient but not as a carbon source.
A “vector” is defined as DNA which can comprise, i.e. carry and maintain the DNA fragment of the invention, including, for example, phages and plasmids. These terms are understood by those of skill in the art of genetic engineering. The term “expression cassette” denotes a nucleotide sequence encoding a pre-protein, operably linked to a promoter and a terminator. As for vectors containing an expression cassette, the terms “vector” and “expression vector” are synonyms.
The term “oligonucleotide” is used for a nucleic acid molecule, DNA (or RNA), with less than 100 nucleotides in length.
“Transformation” means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration.
The term “expression” and the verb “to express” denote transcription of DNA sequences and/or the translation of the transcribed mRNA in a host organism resulting in a pre-protein, i.e. not including post-translational processes.
A nucleotide sequence “encodes” a peptide or protein when at least a portion of the nucleic acid, or its complement, can be directly translated to provide the amino acid sequence of the peptide or protein, or when the isolated nucleic acid can be used, alone or as part of an expression vector, to express the peptide or protein in vitro, in a prokaryotic host cell, or in a eukaryotic host cell.
A “promoter” is a regulatory nucleotide sequence that stimulates transcription. These terms are understood by those of skill in the art of genetic engineering. Like a promoter, a “promoter element” stimulates transcription but constitutes a sub-fragment of a larger promoter sequence.
The term “operably linked” refers to the association of two or more nucleic acid fragments on a single vector so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence, i.e. a nucleotide sequence encoding a protein or a pre-protein, when it is capable of affecting the expression of that coding sequence, i.e., that the coding sequence is under the transcriptional control of the promoter.
The term “polypeptide” or “protein” denotes a polymer composed of more than 90 amino acid monomers joined by peptide bonds. The term “peptide” denotes an oligomer composed of 90 or fewer amino acid monomers joined by peptide bonds. A “peptide bond” is a covalent bond between two amino acids in which the α-amino group of one amino acid is bonded to the α-carboxyl group of the other amino acid.
The term “pre-protein” denotes a primary translation product that is a precursor of a mature protein, i.e. in this case a protein results from post-translational processing of a pre-protein.
The term “post-translational processing” denotes the modification steps a pre-protein is subjected to, in order result in a mature protein in a cellular or extracellular compartment.
A “signal peptide” is a cleavable signal sequence of amino acids present in the pre-protein form of a secretable protein. Proteins transported across the cell membrane, i.e. “secreted”, typically have an N-terminal sequence rich in hydrophobic amino acids about 15 to 30 amino acids long. Sometime during the process of passing through the membrane, the signal sequence is cleaved by a signal peptidase (Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (eds), Molecular Biology of the Cell, fourth edition, 2002, Garland Science Publishing). Many sources of signal peptides are well known to those skilled in the art and can include, for example, the amino acid sequence of the α-factor signal peptide from Saccharomyces cerevisiae and the like. Another example is the bovine signal peptide of the native bovine pancreatic desoxyribonuclease I pre-protein. In general, the pre-protein N-terminus of essentially any secreted protein is a potential source of a signal peptide suitable for use in the present invention. A signal peptide can also be bipartite comprising two signal peptides directing the pre-protein to a first and a second cellular compartment. Bipartite signal peptides are cleaved off stepwise during the course of the secretory pathway. A specific example therefor is the prepro peptide of the α-factor from Saccharomyces cerevisiae (Waters et al., J. Biol. Chem. 263 (1988) 6209–14).
Pre-proteins with an N-terminal signal peptide are directed to enter the “secretory pathway”. The secretory pathway comprises the processes of post-translational processing and finally results in secretion of a protein. Glycosylation and the formation of disulfide bonds are processes that are part of the secretory pathway prior to secretion. In the present document it is understood that proteins secreted by methylotrophic yeast strains have passed through the secretory pathway.
Until presently, a major source of bovine pancreatic desoxyribonuclease I is pancreatic tissue obtained from slaughtered cattle. The enzyme is usually purified from the tissue material using chromatographic separation techniques such as those described by Funakoshi, A., et al., J. Biochem. (Tokyo) 88 (1980) 1113–1138; Paudel, H. K., and Liao, T. H., J. Biol Chem. 261 (1986) 16006–16011; and Nefsky, B., and Bretscher, A., Eur. J. Biochem. 179 (1989) 215–219. The purification process under the conditions of a research laboratory yields a specific activitiy in the range of 1,000 units per mg of protein obtained from 400 g of tissue, as explicitly reported by Paudel, H. K., and Liao, T. H., J. Biol Chem. 261 (1986) 16006–16011. In an upscaled industrial process, the isolation of bovine pancreatic desoxyribonuclease I from bovine pancreatic tissue yields preparations of isolated enzyme which usually exhibit a specific activity of 3,500 units per mg of protein (our own unpublished observation). Commercial preparations of research-grade bovine pancreatic desoxyribonuclease I purified from bovine pancreas usually have a specific activity below this value as exemplified by Roche products (Roche Diagnostics GmbH, Mannheim, Germany; catalogue items as of November 2002) having the catalogue numbers 1284932 (2,000 units/mg), 104132 (3,000 units/mg), 104159 (2,000 units/mg), and Sigma-Aldrich products (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany; catalogue items as of November 2002) having the catalogue numbers D5025 (2,000 units/mg) D4263 (2,000 U/mg), D4513 (2,000 units/mg), DN-25 (400–800 units/mg). Generally, pancreas tissue as source for bovine pancreatic desoxyribonuclease I poses a problem as this tissue has a high content of other digestive pro-enzymes and their activated forms. On the one hand, the mixture from which the desired enzyme is purified is therefore very complex and requires elaborate separation techniques. On the other hand, proteases among the digestive enzymes may destroy the desired protein, especially during the first steps of the purification process when pancreatic tissue is homogenised. One could also speculate that pancreatic tissue homogenate contains inhibitory substances that inactivate a substantial portion of the bovine pancreatic desoxyribonuclease I, thereby limiting the specific activity of active enzyme that can be purified from this source.
It is also known to the art that recombinant expression of an enzymatically active bovine pancreatic desoxyribonuclease I protein in a bacterial host is possible. However, overexpression poses a problem owing to the intrinsic toxicity of an endonuclease for a bacterial cell. Apparently, toxicity is caused by intracellular degradation of host cell DNA with high levels of active bovine pancreatic desoxyribonuclease I enzyme being present in the bacterial cytoplasm. In a bacterial cell transcription and translation are tightly connected as opposed to eukaryotic cells where these processes take place in separate compartments, i.e. the nucleus and the cytoplasm. Thus, the selection of bacterial clones overexpressing bovine pancreatic desoxyribonuclease I at the same time promotes instability of these clones (e.g. genetic instability) and/or the recombinant desoxyribonuclease protein to be produced. Attempts to overcome this problem were mainly based on tightly regulated and inducible bacterial expression systems.
Worrall, A. F., and Connolly, B. A. (J. Biol. Chem. 265 (1990) 21889–21895) expressed an active bovine pancreatic desoxyribonuclease I protein in E. coli making use of a synthetic coding sequence adapted to the codon usage of the host organism. Transcriptional expression was under the control of the λpL promoter. The recombinant protein was produced intracellularly and afterwards solubilised by means of sonication. The recombinant active protein was found to be toxic for the bacterial host stain. Thus, expression yield was in the range between 100 μg to 1 mg/l culture. The specific activity of the recombinant active bovine pancreatic desoxyribonuclease I from E. coli was found to be identical to that of the native protein. The value given in the document (5×108 units/g of protein) was not the true value of the preparation but was corrected for purity. The recombinant bovine pancreatic desoxyribonuclease I protein was purified only partially and the document is completely silent about the specific desoxyribonuclease activity of a substantially pure and/or research-grade product.
Chen, C. Y., et al. (Gene 206 (1998) 181–184) expressed in E. coli a cDNA representing the original bovine pancreatic transcript. The construct was expressed in the strain BL21(DE3)pLysE and transcribed by an IPTG-inducible T7 polymerase. However, overexpression of an active bovine pancreatic desoxyribonuclease I was found to be limited in quantity and the product appeared to be toxic for the bacterial host organism. Due to cell lysis upon induction, bovine pancreatic desoxyribonuclease I activity was found in the supernatant but also in the cellular fraction of the culture. desoxyribonuclease enzyme activity was measured using an assay for enzyme activity that differed from the Kunitz assay in that there are changes with respect to divalent cations and their concentration in the reaction buffer, as well as its pH. However, generally the units detected by this assay appear to be comparable to those of the Kunitz assay. According to the assay of this document, the approximate yield from the induced culture was 3,500 units/l. The specific enzyme activity of the recombinant bovine pancreatic desoxyribonuclease I produced in E. coli was 908 units/mg was in the same range as the specific activity of native bovine pancreatic desoxyribonuclease I purified in parallel from pancreatic tissue (938 units/mg).
As it is commonly observed when post-translationally processed proteins of eukaryotic origin are expressed in a prokaryotic host system, the bovine pancreatic desoxyribonuclease I obtained by means of recombinant expression in E. coli markedly differs from the native protein. Particularly N-glycosylation which is a hallmark of the native pancreatic desoxyribonuclease I protein is absent in the recombinant product derived from prokaryotic expression systems. Glycosylation does not take place in E. coli and the publications by Worrall, A. F., and Connolly, B. A., J. Biol. Chem. 265 (1990) 21889–21895 as well as Chen, C. Y., et al., Gene 206 (1998) 181–184, confirm that the recombinantly produced bovine pancreatic desoxyribonuclease I proteins were not glycosylated. Also, folding of the protein and the formation of disulfide bonds are controlled differently in eukaryotic cells (Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (eds), Molecular Biology of the Cell, fourth edition, 2002, Garland Science Publishing). Above all, the formation of disulfide bridges in the recombinantly produced protein can be assumed to occur randomly once SH groups of the protein come in contact with aerial oxygen.
Glycosylated bovine pancreatic desoxyribonuclease I also was expressed in cultured mammalian cells. Nishikawa, A., et al. (J. Biol. Chem. 272 (1997) 19408–19412) produced bovine pancreatic desoxyribonuclease I as well as human desoxyribonuclease I in COS-1 cells in order to study mannose phosphorylation of these proteins. The document is however completely silent regarding yield or activity of the recombinant desoxyribonuclease I produced. A further glycosylation study on bovine pancreatic desoxyribonuclease I was published by Nishikawa, A., and Mizuno, S., Biochem. J. 355 (2001) 245–248, dealing with the efficiency of N-linked glycosylation of bovine pancreatic desoxyribonuclease I. Also this document is completely silent regarding yield or activity of the recombinant desoxyribonuclease I produced.
Recombinant expression of human pancreatic desoxyribonuclease I in embryonic kidney 293 cells was described by Shak, S., Proc. Natl. Acad. Sci. USA 87 (1990) 9188–9192 as well as by the same author in WO 90/07572. In the latter document it is stated that the specific activity of recombinant human pancreatic desoxyribonuclease from 293 cells appeared to be comparable to that of bovine desoxyribonuclease (Sigma, product D5025) which was used as a reference. According to the supplier's (Sigma) catalogue the product has a specific activity of 2,000 units/mg which puts the recombinantly expressed human pancreatic desoxyribonuclease I protein in the same range. Moreover, according to the invention described in WO 90/07572, recombinant desoxyribonuclease is preferably expressed in mammalian cells but also in prokaryotes, fungi, yeast, pichia, insects and the like. However, apart from expression in mammalian cells the document does not disclose any other example for eukaryotic expression systems. Moreover, the document is completely silent regarding glycosylation, the formation of disulfide bridges and the specific activity of recombinant desoxyribonuclease when produced in other eukaryotic expression systems. Furthermore, the document is completely silent on the impact of secretion on the yield of the desired protein as well as on its activity.
Using a similar expression system as in WO 90/07572, WO 96/26278 describes the production of human desoxyribonuclease I variants that exhibit a reduced binding affinity to actin. EP 1 122 306 discloses the expression of a human desoxyribonuclease II in HeLa cells.
The methods that are provided by the state of the art to produce a bovine pancreatic protein with desoxyribonuclease activity have certain disadvantages. The present invention provides an improved method.
It is an object of the invention to provide a cost-effective alternative source for bovine pancreatic desoxyribonuclease I. It is a further object of the invention to purify bovine pancreatic desoxyribonuclease I as a recombinant protein synthesised by a non-animal host organism. It is another object of the invention to provide an expression system in which the host organism tolerates the recombinant bovine pancreatic desoxyribonuclease I better than bacteria. Another object of the invention is to provide an expression system which simplifies and accelerates the separation of bovine pancreatic desoxyribonuclease I from cellular or media components, therefore conserving enzyme activity which otherwise may be lost. Yet another object of the invention is to provide a production procedure that leads to an enzyme preparation with a high specific activity. Yet another object of the invention is that the production procedure is amenable to upscaling towards a cost-effective industrial process.